Nuclear magnetic resonance spectrometer for liquid-solution

ABSTRACT

In a nuclear magnetic resonance spectrometer, the shape of a detection coil is changed from a conventional cage type to a solenoid type of higher sensitivity. Accordingly, differing from the conventional superconductive magnet of multilayer air core solenoids, a superconductive magnet is right and left divided to split magnets for generating 11 T preferably, 14.1 T in the horizontal direction, and the magnetic field uniformity is set to 0.001 ppm or less and the temporal stability is set to 0.001 ppm or less.

BACKGROUND OF THE INVENTION

The present invention relates to a nuclear magnetic resonancespectrometer suitable for analyzing, in a liquid-solution, the structureand interaction of protein and organic molecules such as substrate andligand interacting with the protein.

A method for analyzing organic matter utilizing nuclear magneticresonance (NMR) has been making rapid progress in recent years. Inparticular, a method has been used in combination with the technique ofstrong superconductive magnets to make it possible to highly efficientlyanalyze the structure of an organic compound such as protein having acomplicated molecular structure on atomic level. The present inventionis directed to a nuclear magnetic resonance (NMR) spectrometer necessaryfor analyzing the structure on atomic level and interaction of proteinmolecules in an aqueous solution dissolving a small quantity of protein,especially, an energy spectrometer differing from a medical MRIdiagnostic apparatus aiming at imaging of a tomogram of human bodyneeding so-called millimeter class image resolution in that performancesuch as the magnetic field intensity being one order or more higher, themagnetic field uniformity being of four order and the stability beingthree order higher is needed, thus requiring quite different designtechnique and apparatus manufacture technique. A detailed description ofa conventional high-resolution NMR spectrometer is given in “NMR forProtein” by Yoji Arata, published by Kyoritu-shuppan, pp. 33-54, 1996.As up-to-date inventions concerning the typical apparatus constructionwhen the NMR is utilized for analysis of protein, one may refer to aninvention relating to a superconductive magnet that shows the typicalconstruction of a multilayer air-core solenoid coil as disclosed inJP-A-2000-147082, an invention relating to a signal detection techniquethat shows a cage type superconductive detection coil as disclosed inU.S. Pat. No. 6,121,776 and examples of signal detection technique basedon a conventional barrel type or cage type coil as disclosed inJP-A-2000-266830 and JP-A-6-237912. According to these reports, all ofthe conventional high-sensitivity NMR spectrometers for protein analysisuse a superconductive magnet unit constructed of solenoid coils used incombination to generate a magnetic field in the vertical directions, anelectromagnetic wave at 400 to 900 MHz is irradiated on a sample and aresonance wave generated from the sample is detected by utilizing thebarrel or cage type detection coil. Also, as shown in an example of U.S.Pat. No. 6,121,776, a contrivance is made to improve the S/N sensitivityratio by utilizing a detector cooled to low temperatures with a view todecreasing heat noise during reception.

Historically, in the high-sensitivity NMR spectrometer, improvements insensitivity have been achieved by a method in which the basicconstituents of a system such as antenna and magnet are kept to remainunchanged but the center magnetic field intensity of the superconductivemagnet is increased. Accordingly, the maximum NMR measurementsensitivity reported till now can be obtained with a NMR spectrometer at900 MHz utilizing a large superconductive magnet having a centermagnetic field of 21.1 tesla and the basic constituents of thespectometer remain unchanged as compared to those in the prior art ofJP-A-2000-147082. In the analysis of protein using a liquid-solution,the improved center magnetic field is effective to clarify separation ofimprovement in sensitivity and chemical shift.

In attaining the effect of improved sensitivity attributable to the formor shape of detection coil, it has been known, for example, as describedin “Book of NMR” by Yoji Arata, published by Maruzen, PP. 325-327, 2000,that the solenoid coil conventionally used as detection coil isadvantageous over the barrel or cage type in various points. Forexample, the solenoid coil is advantageous in easy controllability ofimpedance, filling factor and efficiency of RF magnetic field. But,according to the literature, when the sensitivity is thought much of insuch an application of measuring protein dissolved by a small quantityin an aqueous solution, winding a solenoid coil around a sample tubeplaced vertically to the magnetic field is practically impossible and ingeneral, is not utilized. In an exceptional application where highlysensitive measurement is carried out by using a small quantity of samplesolution, the above technique is limitedly utilized through a methodutilizing a particularly designed micro-sample tube to carry outmeasurement by using a special probe.

Recently, in a special example disclosed in JP-A-11-248810, a method iscontrived according to which a bulky magnet of high-temperaturesuperconductivity is magnetized in the horizontal direction and a NMRsignal is detected with a solenoid coil. Further, JP-A-7-240310discloses a method for constructing a superconductive magnet and acooling container to meet a general NMR application directed toeliminate constraints on the top or ceiling height of apparatus.However, any method of improving the detection sensitivity necessary foranalyzing protein and any method of technically coping with magneticfield uniformity and temporal stability of magnetic field have not beenknown yet.

Recently, with needs for study of protein promoted, needs for analyzinga sample in which protein has a small degree of solubility to water haveincreased and there is a need of improving the sensitivity ofmeasurement of NMR. To adapt the NMR spectrometer to the needs as above,the measurement sensitivity must be improved while maintaining a samplespace comparable to that of the conventional apparatus and besides themaintenance of the stability of a superconductive magnetic field over along time of data integration is indispensable. The improved measurementsensitivity is particularly advantageous in that for samples havingsubstantially the same solubility, not only the measurement time can beshortened but also the sampling amount can be decreased, therebyensuring that the protein of small solubility can be analyzed toadvantage. Accordingly, the NMR spectrometer used for analysis ofprotein is required of far more excellent detection sensitivity andstability than those in the conventional NMR and in addition, isrequired to have ability to detect NMR signals accurately and stablyover a long time of one week or more. This is because if the magneticfield varies during measurement, the peak of NMR signal is caused toshift and especially, in measurement of interaction, the peak shift dueto interaction cannot be discriminated from that due to instability ofthe magnetic field. If the magnetic field is non-uniform, desired peaksoverlap each other, raising a problem that discrimination of interactionis difficult to achieve. Therefore, it should De noticed that, in futureNMR techniques aiming at performing various kinds of analysis ofprotein, development of new technology not lying on mere extension ofthe conventional general NMR spectrometers will be needed.

For example, specification of magnetic field uniformity in the generalNMR spectrometer is 0.01 ppm in a sample space, that is, 0.01 ppm interms of temporal stability. When this value is indicated in terms ofproton NMR for general 600 MHz use, a permissible error of 6 Hz results.In the case of the aforementioned analysis of interaction of protein,however, spatial and temporal resolution of at least 1.0 Hz or less isrequired and preferably, 0.5 Hz or less is needed. In a method capableof implementing the magnetic field stability and the temporal stabilityof magnetic field, the construction of superconductive magnet anddetection coil must be optimized. Accordingly, the performance of theconventional, generally used NMR spectrometer is insufficient and thestability and magnetic field uniformity higher by one order or more thanthose of the conventional spectrometer are required.

In the prior arts, the sensitivity is managed to be improved by relyingon improvements in magnetic field intensity and as a result, theapparatus is increased in size and to cope with problems of leakagemagnetic field and floor strength, there arises a new problem ofinstallation capability such as needs for a dedicated building. Further,disadvantageously, the cost of a superconductive magnet increases. Theimproved sensitivity has an upper limit of about 21 T because ofconstraints due to a critical magnetic field of a superconductivematerial and for more upgraded improvements in sensitivity, the adventof a technique for improving detection sensitivity based on a new meanswithout resort to the magnetic field intensity has been desired.

The aforementioned high-sensitivity measuring method utilizing thesolenoid coil can be used with a special sample tube for a very smallquantity of sample and a special detection probe but it cannot beapplied to analysis based on a general protein solution of about 10 cc.The method for generating a magnetic field in the horizontal directionby means of a strong magnet and detecting NMR signals by means of asolenoid coil as described in the example of JP-A-11-248810 can generateonly a magnetic field of not greater than 10 T at the surface of ahigh-temperature superconductor, with the result that the magnetic fieldat a sample part is about several tesla at the most, thus proving thatthe method of interest cannot generate a magnetic field of 11 tesla ormore necessary for analysis of protein, preferably, a magnetic field of14.1 tesla or more in a desired sample space. Further, in this method,owing to the effect of a magnetic flux creep phenomenon of thehigh-temperature superconductor, the temporal stability 1.0 Hz/hour orless necessary for analysis of protein is substantially difficult toachieve. As regards the magnetic uniformity necessary for analysis ofprotein, non-homogeneity attributable to the manufacture process of ahigh-temperature superconductive bulky material also makes it difficultto attain the magnetic field uniformity within 1.0 Hz in terms of protonNMR frequency in a space defined by 10 mm diameter×20 mm length.

As described above, while a breakthrough technique meeting the needs foranalysis of protein is desired to be developed in connection with theconventional techniques, the advent of a new solving method for furtherimprovements in sensitivity has been desired under the present-daycircumstances that improving the sensitivity based on the magnetic fieldhas reached limits.

For the purpose of conducting an efficient and accurate. Analysis of theinteraction of protein in a liquid-solution with low molecules such assubstrate and ligand, for which needs are considered to increase infuture, it is empirically preferable that a suitable quantity of samplebe measured at 600 to 900 MHz and with a center magnetic field of about14 to 21 T and the measurement sensitivity be increased beyond thepresent one to increase the throughput. Generally, in a spectrometeroperating at 800 MHz or more, for the purpose of making full use of thesuperconductive characteristics to an extreme, operation is carried outby depressurizing liquid helium at 4.2 K and excessively cooling it to1.8 K. Therefore, complexities in apparatus operation are aggravated andmaintenance is laborious. In addition, the magnetic unit Increases insize to increase leakage magnetic field and typically, a dedicatedbuilding is needed. Especially, the leakage magnetic field in thevertical direction increases as the center magnetic field increases inthe conventional system, so that in an apparatus of 900 MHz class, forinstance, a leakage magnetic field occurs extending up to 5 m in theheight direction, and from the viewpoint of apparatus installation,there needs a tall building of high ceiling. As a result, theconstruction cost increases disadvantageously. Further, the conventional900 MHz superconductive magnet is sized such that only a magnet part hasa diameter of 1.86 m and a height of several meters, as described inIEEE. Transactions on Applied Superconductivity, Vol. 10, No. 1, page728-731.

The present invention intends to provide a novel NMR spectrometer inwhich the measurement sensitivity of NMR signals can be increased hit atleast 2.5 times or more of that in the conventional apparatus at about600 MHz (14.1 T) under a condition that a normal sample tube of 5 to 10mm diameter is mainly used and a sample liquid-solution is charged inthe tube up to a height of about 30 mm and the temporal stability andspatial uniformity of a superconductive magnet necessary for analysis ofprotein can be provided. In the construction of the present invention,the operating temperature of a system is not set to 4.2 K. By applyingthe present invention, it is also possible to aim at achieving extremityperformance but depending on applications, operation at the conventionalmagnetic field limit 21.1 T that is, at 900 MHz and at 1.8 K can proceedand in that case, the sensitivity can be improved by 40% of that in theconventional system, proving that overcoming the detection sensitivitylimit attributable to magnetic field intensity, conventionallyunattainable, can succeed for the first time.

SUMMARY OF THE INVENTION

The present inventors have studied earnestly and deeply to find problemscommon to the present-day NMR spectrometers and contrive countermeasuresthereagainst.

To maintain compatibility between costs and installation capability, thepresent-day NMR spectrometer has developed through a method in which aliquid-solution sample is placed in the center of a multilayer air-coredsolenoid coil having excellent magnetic field uniformity and a signal isdetected by a barrel or cage type antenna. Historically, as the NMRtechnology has advanced from a low magnetic field of 400 MHz or lessthanks to the advance of measuring technique and analytical method, themeasuring sensitivity has been improved by strengthening the centermagnetic field while observing the fundamental form. Recently, anexample using a cage type antenna of superconductive type with the aimof reducing heat noise has also been reported. The present inventorshave studied earnestly and deeply a method capable of making the signalintensity far higher than that in the conventional system while keepingthe magnetic field identical to find that this problem can be solvedaccording to features of the invention to be described below.

According to one feature of the invention, the sensitivity is improvedby applying a detection coil of solenoid type under conditions that themagnetic field is 400 MHz or more, preferably, about 600 MHz to 900 MHzsuitable for NMR of a liquid-solution defined by a sample space having adiameter of 5 to 10 mm and a height of 20 mm and the detection coil hasa diameter of 5 to 10 mm and a height of about 20 mm to permit a normalsample tube for NMR research to be used as it is. In principle, it canbe expected that the sensitivity can be improved at least 2.5 times by adifference in shape factor of the detection coil and can be furtherimproved by virtue of other factors, with the result that integratingtime of data can be shortened to 1/10 or less. The liquid-solutionsample is inserted vertically from above into the sample tube of 5 to 10mm diameter up to a height of about 20 to 30 mm. In order to detect NMRsignals with high sensitivity by means of the solenoid coil having itswinding axis in the vertical direction, it is necessary that a magneticfield generated by a superconductive magnet be oriented in thehorizontal direction and the liquid-solution sample be placed removablyin the center of the magnetic field. To this end, the superconductivemagnet must be constructed of right and left divided, paired splitmagnets in contrast to the conventional simplified solenoid magnet.Here, to meet the special analytical utilization of analyzing proteindissolved in the liquid-solution, design and manufacture of thesuperconductive magnet must be optimized to meet the aforementionedtemporal stability of 1.0 Hz/hour in terms of proton NMR frequency andspatial uniformity of 1.0 Hz or less in the sample space. This design ismore stringent by one order or more than the conventional design and isbeyond the range of construction achievable by simply combining theknown techniques. Therefore, each of the split magnets is subjected tosufficient design and study for the purpose of generating a highlyprecise magnetic field of 0.000 ppm that is a limit of effective figureon computers and thereafter optimally arranged and combined with a coilof a superconductive conductor for high magnetic field made of Nb₃Sn forinstance and a superconductive coil for low magnetic field made from aNbTi superconductive conductor. There is no example of constructing ageneral-purpose liquid-solution protein analyzing NMR spectrometer usingthe split magnets. The inventors have studied earnestly and deeply andfound, for the first time in the world, that the temporal stability andspatial stability applicable to the analysis of protein, that is, within1.0 Hz in the sample space and within 1.0 Hz per hour in terms of protonNMR frequency can be accomplished by means of the present spectrometer.A magnet optimizing technique the present inventors have accumulatedearnestly makes it possible to design a uniform magnetic field of acomplicated split coil system, such a design being difficult to achievein the past. It is found that the size of a magnet part including a lowtemperature container can be compacted to be of about 1 m width and 1 mheight per unit to thereby ensure that a space saved, highly integratedexperimental apparatus can be constructed while suppressing the leakagemagnetic field and a liquid-solution NMR spectrometer of high throughputhaving an integrating time of data about 10 times the integrating timein the conventional system can be provided.

More specifically, in a NMR spectrometer for liquid-solution whichcomprises a superconductive magnet, a high-frequency transmission coiland a reception coil and in which a sample dissolved in aliquid-solution is charged in a sample tube up to a height of about 10to 30 mm, the sample tube being inserted vertically from above andhaving a diameter of 5 to 10 mm, a stationary magnetic field generatedby the superconductive magnet is 11 T or more, preferably, 14.1 T ormore, the direction of the magnetic field generated by thesuperconductive magnet is in the horizontal direction, a change per hourof proton NMR frequency due to a change of the stationary magnetic fieldis 1.0 Hz or less, the magnetic field uniformity in a sample space is1.0 Hz or less in terms of proton NMR frequency, the liquid-solutionsample is inserted substantially vertically from above and placed, andthe reception coil is a solenoid coil placed near the magnetic fieldcenter from below the apparatus, whereby a small quantity of proteincharged in the sample tube of 5 to 10 mm diameter can be analyzed withhigh sensitivity.

Further, according to the invention, in a NMR spectrometer whichcomprises a superconductive magnet, a high-frequency transmission coiland a reception coil and in which a sample such as protein dissolved ina liquid-solution is charged in a sample tube up to a height of about 10to 30 mm, the sample tube being inserted vertically from above andhaving a diameter of 5 to 10 mm, a stationary magnetic field generatedby the superconductive magnet is 11 T or more, preferably, 14.1 T ormore, the direction of the magnetic field generated by thesuperconductive magnet is in the horizontal direction, a change per hourof proton NMR frequency due to a change of the stationary magnetic fieldis 1.0 Hz or less, the magnetic field uniformity in a sample space is1.0 Hz in terms of proton NMR frequency, the liquid-solution sample isplaced substantially vertically from above in the magnetic field center,and the reception coil is a solenoid coil made of a superconductivematerial, placed from below the apparatus and cooled to asuperconductivity revealing temperature or less.

Preferably, the organic sample may be a polymer compound, protein orligand.

Preferably, the superconductive magnet is paired split magnets forgenerating a magnetic field in the horizontal direction.

The superconductive magnet may be a toroidal magnet placed horizontally.

Further, according to the invention, in a NMR spectrometer whichcomprises a superconductive magnet, a high-frequency transmission coiland a reception coil and in which a sample such as protein dissolved ina liquid-solution is charged in a sample tube up to a height of about 10to 30 mm, the sample tube being inserted vertically from above andhaving a diameter of 5 to 10 mm, a stationary magnetic field generatedby the superconductive magnet is 11 T or more, preferably, 14.1 T ormore, the superconductive magnet is a toroidal magnet placed in thehorizontal direction, a change per hour of proton NMR frequency due to achange of the stationary magnetic field is 1.0 Hz or less, the magneticfield uniformity in a sample space is 1.0 Hz or less in terms of protonNMR frequency, a plurality of liquid-solution samples are placedcircumferentially of the toroidal coil at intervals of substantiallyequidistance, and the reception coil corresponding to each sample is asolenoid coil made of a superconductive material, placed from below theapparatus and cooled to a superconductivity revealing temperature orless.

Further, the superconductive magnet is a toroidal magnet placedhorizontally and the magnetic field intensity applied to the individualsamples is regulated to clearly discriminate NMR signals generated fromadjacent plural samples from each other.

Structurally, in the liquid-solution NMR spectrometer according to theinvention, the liquid-solution sample is placed from above the apparatusin the magnetic field center, the detection coil is a solenoid coilplaced from below the apparatus in the magnetic field center, thedirection of the magnetic field is horizontal, and the superconductivemagnet is right and left divided.

The present invention is in no way limited to the features set forth sofar and the above and other features will be described in greater detailin the following.

According to the invention, there is provided a novel NMR spectrometerin which the measurement sensitivity of NMR signals can be increased byat least 2.5 times or more of that in the conventional apparatus at 600MHz (14.1 T) under a condition that the normal sample tube of 5 to 10 mmdiameter is mainly used and a sample liquid-solution is charged in thetube up to a height of about 30 mm, and the temporal stability andspatial stability of a superconductive magnet necessary for analysis ofprotein can be provided. Further, when the apparatus is operated at 21.1T, that is, 900 MHz and at 1.8 K, the sensitivity can be largelyimproved in comparison with the conventional apparatus and the detectionsensitivity limit attributable to the magnetic field intensity, whichcannot be overcome conventionally, can be overcome remarkably for thefirst time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an example of construction of a NMRspectrometer system of the invention.

FIG. 2 is a diagram showing an example of construction of an open typeNMR spectrometer system of the invention.

FIG. 3 is a diagram showing an example of construction of a toroidaltape NMR spectrometer system of the invention.

FIG. 4 is a diagram showing an example of construction of a NMR signaldetection system of the invention.

FIG. 5 is a diagram showing an example of construction of a NMRdetection coil system of the invention.

FIG. 6 is a diagram showing an example of construction of a magneticshield in the NMR spectrometer system of the invention.

FIG. 7 is a diagram showing construction of a NMR spectrometer in acomparative example.

FIG. 8 is a diagram showing construction of a detection coil in acomparative example.

FIG. 9 is a diagram showing construction of a NMR system combination ina comparative example.

FIG. 10 is a diagram showing combined construction of a NMR system in acomparative example.

FIG. 11 is a diagram showing combined construction of a NMR system in acomparative example.

FIG. 12 is a diagram showing combined construction of a NMR system in acomparative example.

DESCRIPTION OF THE EMBODIMENTS Embodiment 1

A first embodiment of the present invention is shown in FIG. 1. Ofsuperconductive magnets 1, 2 and 3, an inner one closer to a sample hasa coin made of a material of higher superconductivity critical magneticfield. For example, coils of the superconductive magnets 1, 2 and 3 aremade of Nb₃Al, Nb₃Sn and NbTi, respectively, but as necessary, they mayoptimally be combined with each other to provide a desired value of amagnetic field generated by the coils and a desired value of uniformity.As an example, either a superconductive material of a Bi system such asBi₂Sr₂CaCu₂O₉ or of a Y₁Ba₂Cu₃O₇ or MgB₂ may be used. Thesuperconductive magnets having the coils combined as above generate amagnetic field in the horizontal direction. In FIG. 1, a protein sample4 dissolved in an aqueous solution is charged in a sample tube up to aheight of 30 mm, the sample tube having a diameter of 5 to 10 mm andbeing made of glass possessing magnetic properties equivalent to thoseof water, and is inserted in the magnetic field center from above theapparatus so as to be placed vertically. The magnetic field is appliedto the sample laterally thereof. Accordingly, the superconductivemagnets have each the coil wound solenoidally about a horizontal windingaxis and are each placed in right and left symmetry. The magnets areintegrated or unified compactly to have a maximum width of 400 mm and amaximum height of 700 mm. In the center of the thus integrated magnet,the uniformity of magnetic field is regulated to 0.001 ppm or less, thatis, 0.5 Hz or less in terms of proton NMR frequency and the temporalstability is 0.001 ppm/hour or less, that is, 0.5 Hz/hour or less interms of proton NMR frequency. In this case, a coil for uniformityadjustment may be disposed near the magnetic field center, as necessary.The adjustment may be made by using a conductor in the normaltemperature region or another superconductor in the low temperatureregion or by using the two in combination. For example, when theapparatus is used for NMR at a proton NMR frequency of 600 MHz, thecenter magnetic field is set to about 14.1 T and the magnetic fielduniformity is set to 18 mm sphere, that is, 0.5 Hz or less in terms ofproton NMR frequency. Under this condition, the operating temperature ofthe coil can be set to 4.2 K and pumping of liquid helium is unneeded,thus ensuring easy operation. The sample is inserted vertically.

According to the present system, since the magnetic field is generatedin the horizontal direction and the sample is inserted vertically fromabove, there is no fear of dropping the liquid-solution charged in thetest tube. Structurally, the detection coil is inserted from below andtherefore, a sufficient sample space can be assured, thus making itpossible to make full use of the sample space in the measurement needingthe sensitivity for analysis of protein. Depending on a measurementcondition, the sample may be rotated. Even in case the detection coilsystem is cooled to low temperatures, concentration of the sample andadditives can be changed continuously with the arrangement as above andhence various condition changes can be made easily in the study ofinteraction of protein. For detection of NMR signals, a solenoid coilmade of copper and maintained at normal temperature or a solenoid coilmade of a Y system or MgB₂ and cooled to 10 to 20 K is used andstructurally, it is used as a detection coil 5 for antenna that isinserted in the magnetic field center from below the apparatus so as tobe arranged circumferentially of the glass tube charged with the sampleliquid-solution, whereby a detected signal is transmitted to the outsidethrough a signal cable 6. By virtue of insertion of the detection coilfrom below the apparatus, the sample space can be broadened andvibratory noise from the measurement system to the sample can bereduced. The superconductive magnet is held in the permanent currentmode by means of a permanent current switch 9 and the temporal variationof magnetic field is adjusted to 0.5 Hz or less/hour. Thesuperconductive magnet is dipped in liquid helium 7 so as to be kept atlow temperatures and its outer periphery is covered with liquid nitrogen8, thus forming a double structure serving as a low temperaturecontainer capable of saving consumption of helium. Instead of coolingwith liquid helium, a refrigerator free from problems of vibration, forexample, a pulse tube refrigerator can be used to directly cool thesuperconductive magnet. Reduction of leakage magnetic field around themagnet is important from the viewpoint of installation capability andsafety of the apparatus and structurally, the magnet can be providedwith a magnetic shield meeting an installation condition.

As described above, with the above construction, 600 MHz in terms ofproton NMR frequency can be obtained with the 14.1 T center magneticfield in the present embodiment. In comparison with a comparativeexample of conventional NMR system as shown in FIG. 7, the signal/noiseratio (S/N) can be about 2.5˜2.8 times improved, so that in NMR signalintegrating measurement, data integration time can be speeded up byabout 10 times for the sample dissolved with protein of 20 K molecularweight having the same sample concentration. This is measurement timeperformance comparable to that obtained when the same experiment isobserved with a NMR spectrometer corresponding to 1.5 GHz. By providingthe magnetic shield, interference with other analyzers surrounding theapparatus can be decreased and the density of installation ofinstruments can be promoted to advantage.

In the case of 600 MHz, a 5 gauss line of a leakage magnetic field fromthe apparatus is 2 m distant in the vertical direction and maximally 3 mdistant in the horizontal direction. Through this, the apparatus for NMRdata of the same quality as that obtained at 1.5 GHz can be installedwithout utilizing a special dedicated building.

Embodiment 2

A second embodiment of the invention is shown in FIG. 2. Structurally,the present embodiment is substantially the same as the first embodimentbut the low temperature container is divided in correspondence with theleft and right divisions of superconductive magnet to make the user'sspace utilization releasable. Namely, in contrast to the conventionalhermetical sample space, there is an open space around the samplechamber and therefore, dynamic behavior of protein can be measured by,for example, irradiating light or laser rays on the sample. Since adynamic NMR signal as above can be observed, signal transmission orphotosynthesis of protein, for instance, can be examined. In the case ofa special experiment as above, by cooling the superconductive magnetthrough pumping of liquid helium to operate it at 1.8 k, the apparatuscan be operated at a center magnetic field of about 900 MHz (21.1 T).The detection sensitivity in this case is comparable to that in NMR at2.5 GHz obtained with an apparatus converted into the conventional typeof NMR spectrometer shown in FIG. 7, indicating that this is comparableto an intensive magnetic field of 58.75 T extremely exceeding theconventional critical magnetic field by the superconductive material.Consequently, the detection sensitivity at such a high levelunattainable by the conventional system can be realized with themagnetic field intensity at 2.5 GHz (58.75 T) according to theinvention. In this case, the magnitude of a leakage magnetic field inthe vertical direction of the apparatus according to the presentinvention also differs greatly from that in the conventional NMRspectrometer of 900 MHz class, amounting to 3 m distance in the verticaldirection and maximally 4.5 m distance in the horizontal direction orcoil axis direction.

Embodiment 3

A third embodiment of the invention is shown in FIG. 3. Structurally, inthe present embodiment, 8 pairs of superconductive magnets 11 arearranged toroidally in the horizontal direction with a view to reducingthe leakage magnetic field and enhancing the integration orintensiveness of apparatus. More particularly, 8 NMR spectrometers arejuxtaposed in a single low-temperature container cooled with liquidhelium. Each of the 8 pairs of split magnets can be constructed asdetailed in connection with the first embodiment to generate, forexample, about 600 MHz. Preferably, however, the frequency referenced to600 Hz may be changed by 10 Hz between adjacent NMR spectrometers. Forexample, 610 MHz, 620 MHz, 630 MHz and so on are available. This isadvantageous in that even for the same sample 12, NMR signals of thedifferent spectrometers can advantageously be discriminated from eachother. With this construction, since the intensiveness of the NMRspectrometers highly integrated in a space of a diameter of 5 m to 10 mcan be ensured and therefore, a highly economical NMR spectrometersystem of high throughput, reduced in costs of installation andmaintenance, can be provided.

Embodiment 4

A fourth embodiment of the invention is shown in FIG. 4. In the presentembodiment, an example of construction of a NMR system of the inventionwill be described. For supply of liquid-solution sample, an automaticsupply unit 13 such as flow cell is used to continuously provide desiredconcentration, a substrate, ligand or additives such as metal elements.In this manner, a system suitable for study of interaction of proteincan be constructed. A NMR signal is sent through an amplifier 14 to ananalyzer 15 controllable by a workstation 16. For acquisition of NMRsignals, various kinds of pulse sequence are applicable. As necessary, apulse is transmitted and the pulse sequence can be combined with agradient magnetic field. For reduction of heat noise of signal, theamplifier 14 cooled to nearly liquid nitrogen temperature can be used.

Embodiment 5

A fifth embodiment of the invention is shown in FIG. 5. Illustrated inFIG. 5 are details of construction of a detection probe of theinvention. A sample 4 is protein dissolved in an aqueous solution. Thesample 4 is inserted in a glass tube of 5 to 10 mm diameter havingproperties equivalent to those of water and is placed from below theapparatus in the magnetic field center. A detection coil 17 in the formof a solenoid is made of a material of copper or superconductor. In thecase of the superconductor, the detection coil is cooled with helium gas25 to about 10 to 20 K. In the case of copper conductor, the detectioncoil is maintained at normal temperature. The sample may be rotated asnecessary.

Coils 19, 20, 21, 22, 23 and 24 for three-axes gradient magnetic fieldare used in combination to apply a desired magnetic field gradient in adesired direction and they are combined with the pulse sequence so as tobe utilized for analysis. A detected signal is sent to a pre-amplifierby a lead line 18.

According to the present invention, the sensitivity can be improvedwithout resort to the detection coil using a superconductor, thusattaining the effects that the maintenance capability can be excellentand applicability to the light irradiation experiment described inconnection with embodiment 2 can be permitted. In addition, by makingthe detection coil solenoidal and superconductive, the sensitivity canbe improved by 2.5 times or more of that in the conventional apparatusand for example, when 900 MHz (21.1 T) is applied, the detectionsensitivity corresponding to the conventional 2.5 GHz (58.75 T) can beobtained to ensure that the detection sensitivity unattainableconventionally can be realized to advantage.

Embodiment 6

A sixth embodiment of the invention is shown in FIG. 6. The fundamentalconstruction is the same as that in embodiment 2 of the invention. Toreduce the leakage magnetic field, a combination with a magnetic shieldmade of iron is employed. By virtue of the combination with a returnyoke 26 using iron, the leakage magnetic field to surrounding canadvantageously be reduced to within 2 m distance. As necessary, anactive shield utilizing a superconductive magnet can be used as themagnetic shield. This is meritorious in that in comparison with iron,weight can be reduced. In addition, iron may be used in combination withthe active shield.

COMPARATIVE EXAMPLE 1

As an example of NMR spectrometer construction comparative with that ofthe present invention, the construction of a NMR spectrometer of 600 MHzclass is shown in FIG. 7. The center magnetic field is 14.1 T.Illustrated are a cage-shaped reception coil 27 and a multiplayerair-core solenoid of superconductive magnets 28, 29 and 30. In order toimprove the sensitivity equivalently to the present invention with thisconventional detection method, there is nothing but only a way toincrease the center magnetic field. At present, however, 21 T is a limitand for the same frequency (center magnetic field intensity), thedetection sensitivity is about ⅓ inferior to that of the presentinvention. The uniformity and temporal stability of the magnetic fieldare lower than 0.5 Hz that is necessary for analysis of protein.

COMPARATIVE EXAMPLE 2

As an example of NMR spectrometer construction comparative with thepresent invention, the construction of a cage probe 27 is shown in FIG.8. A barrel coil may sometimes be used but in a method in which it iscombined with the comparative example 1, the same detection sensitivityas that in embodiment 1 of the invention cannot be obtained. In order toimprove the sensitivity with this conventional method, there is nothingbut a way to decrease the temperature. At present, a method of utilizingsuperconductivity has been proposed but problems of increased costs andof maintenance capability are encountered. Further, the detectionsensitivity is about ⅓ inferior to that obtained with the solenoid typedetection coil of the present invention.

COMPARATIVE EXAMPLE 3

An example comparative with the present invention is shown in FIG. 9. Inthis example, a general NMR solenoid coil for generating a uniformmagnetic field in the horizontal direction is used to provide ahorizontal magnetic field type arrangement and is used in combinationwith the cage type detection coil in comparative example 2, whereby atest tube charged with an aqueous solution of protein is inserted in ameasuring portion via a horizontal communication hole. A NMR signal atabout 400 MHz is obtained by a magnetic field at the measuring portionbut in a sample space having a diameter of 10 mm and a length of 20 mm,spatial non-uniformity of magnetic field corresponding to an error of 4Hz is caused. Further, non-uniformity of magnetic field per hour is 3Hz/hour. These values are standard ones in the case of the general NMRspectrometer but they are insufficient for analysis of interaction ofprotein aimed at by the present invention. In addition, duringmeasurement, the solution liquid level in the test tube moves in thehorizontal direction and stable measurement often fails to proceed.

COMPARATIVE EXAMPLE 4

An example comparative with the present invention is shown in FIG. 10. Ageneral NMR solenoid coil for generating a uniform magnetic field isused to provide a horizontal magnetic field type arrangement and ispassed through a communication hole from above the apparatus to reach ameasuring portion so as to be combined with the cage type detection coilof comparative example 2, and an aqueous solution of protein is insertedin a test tube. Since the measuring instrument is inserted from above,the apparatus is increased in size by 200 mm in terms of the maximumdiameter of the communication hole. As a result, the center magneticfield intensity is decreased. The magnetic field intensity is adjustedearnestly but a NMR signal only up to 300 MHz can be obtained in themagnetic field center. Further, in the sample space of 10 mm diameterand 20 mm length, there results magnetic non-uniformity corresponding toan error of 5 Hz. These values are standard ones for the general NMRspectrometer but insufficient for interaction analysis of protein.

COMPARATIVE EXAMPLE 5

An example comparative with the present invention is shown in FIG. 11.Two general NMR solenoid coils of 18 T class for generating a uniformmagnetic field are employed in the vertical direction so as to be usedin combination with the horizontal magnetic field type arrangement.These two coils are combined with the solenoid type detection coil ofthe present invention that is passed from above the apparatus through acommunication hole of 70 mm diameter to reach a measuring portion. Anaqueous solution of protein is inserted in a test tube. All of measuringinstruments and liquid-solution sample are inserted from above. As aresult, the effective space of the sample is narrowed, amounting to adiameter of 1 mm and a length of 20 mm. With this construction, thevolume of the sample can be only 1/25 to 1/100 in comparison with thecase of the sample tube of a diameter of 5 to 10 mm used for the normalliquid-solution NMR spectrometer and the signal intensity from thesample is decreased to 1/25 to 1/100, with the result that in spite ofthe targeted improvements in sensitivity, the sensitivity is converselydegraded to a great extent. On the other hand, the maximum magneticfield of each magnet is 18 T in the center axis direction of the twomagnet systems. Further, the temporal stability of magnetic field isabout 0.001 ppm/h. But the stationary magnetic field intensity in thesample space is 7.5 T and the uniformity of the magnetic field in thesample space is 100 ppm or less, proving that the comparative example ison a level not utilizable for measurement by serving as NMR usable forprotein analysis.

COMPARATIVE EXAMPLE 6

An example comparative with the present invention is shown in FIG. 12.In this example, a solenoidally wound detection coil 31 combined with ahigh-temperature superconductive bulky magnet 32 for generating amagnetic field in the vertical direction is used for NMR measurement ofan aqueous solution of protein. The solenoid coil having a windingextensively protruding from the magnetic field uniformity area ischaracteristic of this example, whereby changes of magnetic flux over awide range can be fetched on the one hand but on the other hand, themeasurement accuracy is inferior to that obtained when the detectioncoil is arranged in a uniform magnetic field. With the bulky magnet, themaximum magnetic field of the magnet is 10 T but the maximum magneticfield in the sample space is 4 T. The magnetic field uniformity isdegraded by non-uniformity of the high-temperature superconductor,amounting to 200 to 500 ppm or less. In addition, the temporal stabilityof magnetic field is affected by a magnetic flux creeping phenomenon ofthe high-temperature superconductor, amounting to about 20 to 100 ppm/h.These values prove that the comparative system cannot at all be used formeasurement in the form of NMR usable for protein analysis.

As has been described, with the recent advance of protein study, needsfor structure analysis of complicated compounds of large molecularweight have been encouraged. Under the circumstances, performancerequired for NMR has been upgraded year by year and the center magneticfield of NMR has been increased for the sake of improving the detectionsensitivity. In principle, the detection sensitivity increases inproportion to the 7/4-th power of the magnetic field but in practice, itis improved in almost direct proportion to the magnetic field. With theexisting superconductive materials, improvements in sensitivity reach alimit and the advent of new detection method and apparatus independentof the magnetic field intensity has been sought. According to theinvention, in the liquid-solution NMR for protein analysis, thedetection sensitivity can be improved by at least 2.5 times of that inthe conventional apparatus without increasing the center magnetic fieldintensity. In the method meeting this effect, the liquid-solution sampleis placed in the magnetic field center from above the apparatus, thedetection coil in the form of a solenoid coil is placed there from belowthe apparatus and the magnetic field direction is in the horizontaldirection in terms of the proton NMR frequency necessary for proteinanalysis, the magnetic field has temporal stability of 1.0 Hz or lessper hour and uniformity of 1.0 Hz or less in the sample space. Thesuperconductive magnet is divided into right and left split magnets. Inother words, the shape of the detection coil is changed from theconventional cage type 27 to the solenoid type 4 having highersensitivity. Accordingly, the superconductive magnet differs from theconventional multiplayer air-core solenoids 28, 29 and 30 in that it isconstructed of split magnets based on superconductive magnets 1, 2 and 3that are right and left divided for generating 11 T, preferably, 14.1 Tin the horizontal direction. The magnetic field uniformity is set to0.001 ppm or less and the temporal stability is set to 0.001 ppm orless. By making the detection coil the solenoid type, the measurement ofhigh sensitivity that is 1.4 times the conventional sensitivity can beensured for the same center magnetic field (resonance frequency) toattain high throughput and the conventionally unattainable NMR detectionsensitivity corresponding to 2.5 GHz or 58.75 T can be realized at 900MHz (21.1 T).

The present invention permits not only the analysis of inter-proteininteraction but also the NMR measurement of super-high sensitivity thatis higher by 40% of the conventional sensitivity. For example, when theapparatus is operated at a center magnetic field of 900 MHz (21.1 T)that is the highest level attainable by the present-daysuperconductivity technology, the detection sensitivity corresponding tothe conventional 2.5 GHz (58.75 T) can be obtained, whereby thedetection sensitivity of super-high sensitivity that is unattainable bythe conventional sensitivity improving method relying on the magneticfield intensity can be reached for the first time to advantage.

Further, the present invention is suitable for protein interactionscreening study. Conceivably, competition for structure analysis ofprotein will grow more intensively in the future post-genome age andwhen the age of utilizing protein clarified in its structure comes,needs for the known interrelation of protein, that is, the interactionscreening are expected to be highlighted. More particularly, theinteraction screening will be applied widely to the field of tailor-mademedicine creation, biotechnology industry, foods and medical treatment.Under a condition that the analysis of stereoscopic structure of proteinwill have advanced in the 5 to 10 years later forthcoming future, newmedicine development positively making advantage of the structureinformation (so-called medicine creation study) will probably make anadvance. In the age for new medicine development as above, the NMRmeasurement technique of the present invention can be utilized toefficiently promote finding of an unknown interaction between proteinknown about its structure and ligand or a low molecular compound. Fornew medicine creation, through a molecular design support combined withcalculation simulation based on a new interaction found by NMR,optimized new medicine development will be advanced by making full useof the high-biotechnology. Through the process as above, costs andperiod for developing a new medicine effectively acting on the humanbody can be shortened drastically. The spread influence as above has theunfathomable spread influence upon Japan and worldwide mankind. From thetechnical point of view, the NMR technique of the present invention canimprove the detection sensitivity by 2.5 times or more of theconventional detection sensitivity and therefore, the integrating timecan be shortened to 1/10. Accordingly, in addition to the aforementionedinteraction detection, the influence of a small quantity of metal uponthe human body can be studied efficiently. As a concrete example, thereis a possibility that the present invention can be applied todevelopment of a remedy for many kinds of diseases affected by a smallquantity of elements and protein in vivo, for example, Alzheimer diseaseand an early diagnosis of chronic/obstinate disease before an attack(diabetes, Creutzfeldt-Jakob disease and the like) by pursuing, on realtime base, the influence of metal elements, of substantially the sameconcentration as that in vivo, upon a existence state of protein and thedynamic state of protein and a small quantity of elements that aremarked in vivo. Further, the present measuring technique can improvemeasuring instruments in maintenance and installation capabilitydrastically and therefore, it is very meritorious to introduce thepresent measuring technique. Especially, high-quality data approximatingthat obtained with a large-scale experimental apparatus of 900 MHz classcan be obtained with relatively small-scale experimental equipments.

It should be further understood by those skilled in the art that theforegoing description has been made on embodiments of the invention andthat various changes and modifications may be made in the inventionwithout departing from the spirit of the invention and the scope of theappended claims.

1-11. (canceled)
 12. A nuclear magnetic resonance spectrometercomprising: a solenoid type superconductive magnet having a horizontalwinding axis, the superconductive magnet symmetrically arranged withrespect to the horizontal winding axis; a low temperature container forcooling the superconductive magnet; and a solenoid type reception coilfor receiving a signal from a sample; wherein the low temperaturecontainer includes a first passage extending along a directionperpendicular to the horizontal winding axis for inserting the sampleand a second passage extending along a direction parallel to thehorizontal winding axis, and wherein the superconductive magnet isoperated in a permanent current mode.
 13. A nuclear magnetic resonancespectrometer according to claim 12, further comprising a magnetic shieldcombined with the low temperature container.
 14. A nuclear magneticresonance spectrometer comprising: a split superconductive magnet havinga horizontal winding axis, the split superconductive magnet includingtwo magnets opposing each other, each magnet comprising a solenoid typecoil having the horizontal winding axis; a low temperature container forcooling the split superconductive magnet; and a solenoid type receivercoil configured to acquire a signal from a sample located in a samplespace; wherein the low temperature container includes a first passageextending along a direction perpendicular to the horizontal windingaxis, and a second passage extending along a direction parallel to thehorizontal winding axis to access the sample space, and wherein thesplit superconductive magnet is operated in a permanent current mode.15. A nuclear magnetic resonance spectrometer according to claim 14,further comprising a magnetic shield combined with the low temperaturecontainer.
 16. A nuclear magnetic resonance spectrometer for aliquid-solution which comprises: a superconductive magnet includingpaired split magnets for generating a magnetic field, a low temperaturecontainer for cooling the superconductive magnet, the low temperaturecontainer having a first space perpendicular to the magnetic field and asecond space along the magnetic field; and a receiver coil disposed toacquire a signal from a sample, wherein the magnetic field is 11 T ormore, variation of proton nuclear magnetic resonance frequency due tovariation of the magnetic field is 1.0 Hz/hour or less, the sample isinserted in the magnetic field from the first space, and the receivercoil is a solenoid coil inserted in the magnetic field, and wherein thesuperconductive magnet is operated in a permanent current mode.
 17. Anuclear magnetic resonance spectrometer according to claim 16, whereinthe receiver coil is made of a superconductive material.